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US 9,066,870 B2Granted
Methods for drug delivery comprising unfolding and folding proteins and peptide nanoparticles
AssigneesNanjing University, Nanjing Effect Pharm Drug Development Corporation
InventorsYiqiao Hu, Jinhui Wu, Dawei Ding, Guangming Gong, Xiaolei Tang, Chunhui Tong, Yan Zhu, Shaoling Li
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Abstract
The present invention provides preparation methods of protein nanoparticles for in vivo delivery of pharmacologically active agents, wherein said methods are to encase pharmaceutically active agents into proteins or peptides to form nanoparticles by unfolding the protein, and subsequently refolding or assembling the protein to produce a pharmacologically active agent encased within a protein nanoparticle.
Claims25
A method for the preparation of nanoparticles fully encasing a pharmacologically active agent within a protein which has been unfolded and subsequently refolded or assembled for in vivo delivery, wherein said method comprises the following steps:
- (a) preparing or obtaining a protein solution comprising said protein in a first solvent;
- and
- (b) contacting said pharmacologically active agent with said protein solution of step (a) in the presence of an unfolding solution, thereby causing unfolding of said protein, and subsequent refolding or assembling of said protein to fully encase said pharmacologically active agent within said protein,
- wherein the pharmacologically active agent is selected from the group consisting of:
- paclitaxel, docetaxel, gambogic acid, doxorubicin, rapamycin, tacrolimus, and combinations thereof.
2 more elements
A method for the preparation of nanoparticles fully encasing a hydrophobic pharmacologically active agent within a protein which has been unfolded and subsequently refolded or assembled for in vivo delivery, wherein said method comprises the following steps:
- (a) preparing or obtaining a protein solution comprising said protein in a first solvent at a temperature ranging from about −20° C. to about 100° C., and a pH ranging from about 3 to about 9;
- and
- (b) contacting said hydrophobic pharmacologically active agent with said solution of step (a) in the presence of an unfolding solution, thereby causing unfolding of said protein, and subsequent refolding or assembling of said protein to fully encase said pharmacologically active agent within said protein;
- wherein said nanoparticles have a diameter ranging from about 5 nm to about 2000 nm and said nanoparticles comprise about 1% to about 40% of pharmacologically active agent by total weight of the nanoparticle;
- wherein said hydrophobic pharmacologically active agent is selected from the group consisting of:
- paclitaxel, docetaxel, doxorubicin, gambogic acid, tacrolimus, rapamycin, and combinations thereof;
- wherein said protein is selected from the group consisting of:
- albumin, transferrin, insulin, endostatin, hemoglobin, myoglobin, lysozyme, immunoglobulins, α-2-macroglobulin, fibronectin, lamin, collagen, gelatin, artificial peptides and proteins, and combinations thereof;
- wherein said first solvent is selected from the group consisting of:
- water, saline, phosphate, acetate, glycine, tris(hydroxymethyl)aminomethane, aqueous solutions containing hydrogen peroxide, aqueous solutions comprising glutathione, sodium chloride solutions, glucose solutions, mycose, mannitol, sucrose, acetyltryptophan, sodium caprylate and combinations thereof;
- and
- wherein said unfolding solution is selected from the group consisting of:
- water, saline, phosphate, acetate, glycine, tris(hydroxymethyl)aminomethane, aqueous solutions containing hydrogen peroxide, aqueous solutions comprising glutathione, sodium chloride solutions, glucose solutions, methanol, ethanol, propanol, isopropanol, formaldehyde, chloroform, acetone, hydrogen sulfide, 2-mercaptoethanol, dithiothreitol, guanidine, urea, perchlorate, tri-n-butylphosphine, captopril, performic acid, penicillamine, glutathione, methimazole, acetylcysteine, 3,3″,3″-phosphinidynettripropionic acid, and combinations thereof.
10 more elements
A method for the preparation of nanoparticles fully encasing paclitaxel within a protein which has been unfolded and subsequently refolded or assembled for in vivo delivery, wherein said method comprises the following steps:
- (a) preparing or obtaining a protein solution comprising said protein in a first solvent at a temperature ranging from about 55° C. to about 75° C., and a pH ranging from about 6 to about 8;
- (b) contacting said paclitaxel with said protein solution of step (a) in the presence of an unfolding solution, thereby causing unfolding of said protein, and subsequent refolding or assembling of said protein to fully encase said paclitaxel within said protein;
- (c) performing dialysis or filtration to remove excess chemicals or to obtain a more concentrated solution;
- and
- (d) subjecting said concentrated solution of nanoparticles of step (c) to a dehydration process to produce preserved nanoparticles capable of incorporation into a pharmaceutical dosage form;
- wherein said nanoparticles have an average diameter ranging from about 50 nm to about 300 nm and said nanoparticles comprise about 1% to about 40% of paclitaxel by total weight of the nanoparticle;
- wherein said unfolding solution is selected from the group consisting of water, saline, phosphate, acetate, glycine, tris(hydroxymethyl)aminomethane, sodium chloride solution, ethanol, acetone, hydrogen sulfide, 2-mercaptoethanol, urea, and combinations thereof;
- and
- wherein said protein is selected from the group consisting of albumin, transferrin, insulin, endostatin, and hemoglobin.
6 more elements
A method for the preparation of nanoparticles fully encasing docetaxel within a protein which has been unfolded and subsequently refolded or assembled for in vivo delivery, wherein said method comprises the following steps:
- (a) preparing or obtaining a protein solution comprising said protein in a first solvent at a temperature ranging from about 55° C. to about 75° C., and a pH ranging from about 6 to about 8;
- (b) contacting said docetaxel with said protein solution of step (a) in the presence of an unfolding solution, thereby causing unfolding of said protein, and subsequent refolding or assembling of said protein to fully encase said docetaxel within said protein;
- (c) dialyzing or filtering said nanoparticles to remove excess chemicals to obtain a more concentrated solution;
- and
- (d) subjecting said concentrated solution of nanoparticles of step (c) to a dehydration process to produce preserved nanoparticles capable of incorporation into a pharmaceutical dosage form;
- wherein said nanoparticles have an average diameter ranging from about 50 nm to about 300 nm and said nanoparticles comprise about 1% to about 40% of docetaxel by total weight of the nanoparticle;
- wherein said unfolding solution is selected from the group consisting of water, saline, phosphate, acetate, glycine, tris(hydroxymethyl)aminomethane, sodium chloride solution, ethanol, acetone, hydrogen sulfide, 2-mercaptoethanol, urea, and combinations thereof;
- and
- wherein said protein is selected from the group consisting of albumin, transferrin, insulin, endostatin, and hemoglobin.
6 more elements
A nanoparticle comprising a pharmacologically active agent fully encased within a protein which has been unfolded and subsequently refolded or assembled, wherein said nanoparticle is suitable for in vivo delivery, and wherein said nanoparticle is produced according to the following steps:
- (a) preparing or obtaining a protein solution comprising said protein in a first solvent at a temperature ranging from about −20° C. to about 100° C., and a pH ranging from about 3 to about 9;
- and
- (b) contacting said hydrophobic pharmacologically active agent with said protein solution of step (a) in the presence of an unfolding solution, thereby causing unfolding of said protein, and subsequent refolding or assembling of said protein to fully encase said pharmacologically active agent within said protein;
- wherein said nanoparticles have a diameter ranging from about 5 nm to about 2000 nm and said nanoparticles comprise about 1% to about 40% of pharmacologically active agent by total weight of the nanoparticle;
- wherein said hydrophobic pharmacologically active agent is selected from the group consisting of:
- paclitaxel, docetaxel, doxorubicin, gambogic acid, tacrolimus, rapamycin, and combinations thereof;
- wherein said protein is selected from the group consisting of:
- albumin, transferrin, insulin, endostatin, hemoglobin, myoglobin, lysozyme, immunoglobulins, α-2-macroglobulin, fibronectin, lamin, collagen, gelatin, artificial peptides and proteins, and combinations thereof;
- wherein said first solvent is selected from the group consisting of:
- water, saline, phosphate, acetate, glycine, tris(hydroxymethyl)aminomethane, aqueous solutions containing hydrogen peroxide, aqueous solutions comprising glutathione, sodium chloride solutions, glucose solutions, and combinations thereof;
- and
- wherein said unfolding solution is selected from the group consisting of:
- water, saline, phosphate, acetate, glycine, tris(hydroxymethyl)aminomethane, aqueous solutions containing hydrogen peroxide, aqueous solutions comprising glutathione, sodium chloride solutions, glucose solutions, methanol, ethanol, propanol, isopropanol, formaldehyde, chloroform, acetone, hydrogen sulfide, 2-mercaptoethanol, dithiothreitol, guanidine, urea, perchlorate, tri-n-butylphosphine, captopril, performic acid, penicillamine, glutathione, methimazole, acetylcysteine, 3,3″,3″-phosphinidynettripropionic acid, and combinations thereof.
10 more elements
A nanoparticle comprising paclitaxel fully encased within a protein which has been unfolded and subsequently refolded or assembled, wherein said nanoparticle is suitable for in vivo delivery, and wherein said nanoparticle is produced according to the following steps:
- (a) preparing or obtaining a protein solution comprising said protein in a first solvent at a temperature ranging from about 55° C. to about 75° C., and a pH ranging from about 6 to about 8;
- (b) contacting said paclitaxel with said protein solution of step (a) in the presence of an unfolding solution, thereby causing unfolding of said protein, and subsequent refolding or assembling of said protein to fully encase said paclitaxel within said protein;
- (c) dialyzing or filtering said nanoparticles to remove excess chemicals to obtain a more concentrated solution;
- and
- (d) subjecting said concentrated solution of nanoparticles of step (c) to a dehydration process to produce preserved nanoparticles capable of incorporation into a pharmaceutical dosage form;
- wherein said nanoparticles have an average diameter ranging from about 50 nm to about 300 nm and said nanoparticles comprise about 1% to about 40% of paclitaxel by total weight of the nanoparticle;
- wherein said unfolding solution is selected from the group consisting of water, saline, phosphate, acetate, glycine, tris(hydroxymethyl)aminomethane, sodium chloride solution, ethanol, acetone, hydrogen sulfide, 2-mercaptoethanol, urea, and combinations thereof;
- and
- wherein said protein is selected from the group consisting of albumin, transferrin, insulin, endostatin, and hemoglobin.
6 more elements
A nanoparticle comprising docetaxel fully encased within a protein which has been unfolded and subsequently refolded or assembled, wherein said nanoparticle is suitable for in vivo delivery, and wherein said nanoparticle is produced according to the following steps:
- (a) preparing or obtaining a protein solution comprising said protein in a first solvent at a temperature ranging from about 55° C. to about 75° C., and a pH ranging from about 6 to about 8;
- (b) contacting said docetaxel with said protein solution of step (a) in the presence of an unfolding solution, thereby causing unfolding of said protein, and subsequent refolding or assembling of said protein to fully encase said docetaxel within said protein;
- (c) dialyzing or filtering said nanoparticles to remove excess chemicals to obtain a more concentrated solution;
- and
- (d) subjecting said concentrated solution of nanoparticles of step (c) to a dehydration process to produce preserved nanoparticles capable of incorporation into a pharmaceutical dosage form;
- wherein said nanoparticles have an average diameter ranging from about 50 nm to about 300 nm and said nanoparticles comprise about 1% to about 40% of docetaxel by total weight of the nanoparticle;
- wherein said unfolding solution is selected from the group consisting of water, saline, phosphate, acetate, glycine, tris(hydroxymethyl)aminomethane, sodium chloride solution, ethanol, acetone, hydrogen sulfide, 2-mercaptoethanol, urea, and combinations thereof;
- and
- wherein said protein is selected from the group consisting of albumin, transferrin, insulin, endostatin, and hemoglobin.
6 more elements
Dependencies are inferred from the claim text.